Cpc100

Isolated small EVs were characterized by their size, concentration and stability (zeta potential) using a tunable resistive pulse sensing (TRPS) technique from qNano (Izon Science, Christchurch, New Zealand). The analysis was performed using NP100 nanopores, capable of detecting particles within the size range of 50–300 nm (as stated by the manufacturer, Izon Science), and compared to the calibration beads CPC100 (mode diameter, 100 nm). Prior to the analyses, sEV samples were diluted 1:1 in 2x PBS supplemented with 0.1% Tween 20, as recommended by the manufacturer. Measurements were made with 44.29 mm of appropriate stretch and 0.44-volt (V) potential applied on the pores, with at least 500 particles being detected. Each measurement was run with a baseline current of 100 nanoamperes (nA) ± 10 nA. All samples were vortexed for 30 s and sonicated for 2 min prior to analysis.
The charges were quantified by a four-calibration point using CPC100 as described [57 (link),58 (link),59 (link)].

The concentration and size distribution of the obtained samples were determined by qNano (Izon Science, Christchurch, New Zealand). First, all samples were serially diluted (1 : 100, 1 : 1000, 1 : 10 000) and analyzed starting at the lowest dilution by NP100 (size range: 50–330 nm) and NP400 (185–1100 nm) nanopores stretched between 45 and 48 mm. Only measurements with a particle count > 500 or a time period of 5 min, linear particle rate in time, and noise below 15 pA were recorded. The pressure was adjusted to achieve a particle flow rate > 100/s and a stable current between 120 and 150 nA. Calibration was performed using calibration beads of a known concentration and size [CPC100 (110 nm) and CPC400 (340 nm), both from Izon Science] diluted at 1 : 1000 according to the manufacturer’s protocol. All samples, including calibration samples, were vortexed for 30 s before obtaining measurements. Data were analyzed by control suite V3.3 software for qNano (Izon Science).

Tunable resistive pulse sensing was conducted using a qNano instrument (Izon) to determine the diameter of the isolated vesicles or particles in solution. More specifically, samples were resuspended in PBS + 0.025% Tween 20 prior to measurement to reduce aggregation. EVs were driven through a qNano size-tunable nanopore (NP100, Izon) and detected one at a time as a transient change in the ionic current flow, which was denoted as a blockade event, with its amplitude representing the blockade magnitude. Because the blockade magnitude is proportional to the particle size, accurate particle sizing was achieved after calibration with particles of a known size (CPC100, Izon) using identical settings. Data processing and analysis were carried out using Izon Control Suite software v3.0 (Izon).

The qNano Gold instrument (Izon Science, Christchurch, New Zeeland) was employed to measure the size distribution and concentration of the isolated NPs using the tunable resistive pulse sensing (TRPS) principle as already reported [53 (link),54 (link)]. Briefly, 35 μL of purified particles were analyzed with a qNano Gold instrument using a NP200 Nanopore (Izon Science) and applying 49 mm stretch, 0.1 V, and 20 mBar parametric conditions. The calibration particles (CPC100, Izon Science) were assayed before the experimental samples under identical conditions. Size and concentrations (2000 events each) were finally determined using the qNano software provided by Izon Science (Izon Control Suite version 3.1).
Zeta potential was evaluated by a Litesizer 500 Particle Analyzer (Anton Paar, Turin, Italy) in aqueous suspension.
DSC and FT-IR analyses on BBR-NPs were performed with the same experimental conditions used to characterize the BBR salts (see Section 2.2).
TEM ultrastructural analysis of NPs was carried out using a Zeiss EM900 electron microscope (Zeiss) operating at 80 kV.

A LM10/14 Nanosight (Nanosight, Malvern) instrument was used to analyse EVs. Prior to analysis, 1:10 dilution of CPC100 (IZON) and 1:1000 dilution of 200 nm polystyrene (Malvern) nanoparticles were used to test the sensitivity of the instrument. EV samples were used at 1:500 dilution. Automatic settings were applied for the minimum expected particle size, minimum track length and blur settings. For capture settings, screen gain was set at 1 and camera level was set at 10 (shutter 1500; gain 680). For analysis settings, screen gain was set at 10 and detection threshold was set at 10. Five movies of 60 s were captured at 30 frames per second for each sample. Data processing and analysis of particle size distribution and concentration were performed using NTA Software (https://www.malvern.com). NTA concentration estimation is dependent on the refractive index of particles under analysis according to the Rayleigh approximation ${\sigma }_s=\frac{2{\pi }^5}{3}\frac{d^6}{{\lambda }^4}{\left(\frac{n^2-1}{n^2+2}\right)}^2$ (where d is the particle diameter, λ is the wavelength, and n is the ratio of particle refractive index to solvent refractive index [17 (link)]), which is known to vary in EV samples due to heterogenic size and content [11 ]. Therefore, NTA analysis was used only to determine PSD but not EV concentration.

The size and concentration of EVs were measured using a tunable resistive pulse sensing (TRPS) method (qNano, Izon Science). Samples were filtered before processed in the qNano instrument using a 1-μm syringe filter (Tisch Scientific, North Bend, OH). 45 μL of biofluid was pipetted into different nanopore stretchable membranes (NP150, NP300, NP600, NP800, and NP1000 from Izon Science) to cover the wide size range of EVs^{36 (link)}. A pressure of 1 kPa and different voltages (0.38 V, 0.32 V, 0.26 V, 0.18 V and 0.12 V) were applied. Polystyrene nanoparticles of different known sizes and concentrations were used for calibration (CPC70, 70 nm; CPC100, 100 nm; CPC200, 200 nm; CPC400, 400 nm; CPC800, 800 nm; CPC1000, 1000 nm from Izon Science, Medford, MA). EVs with a size range of 70–1000 nm were characterized.

Extracellular vesicle concentration was determined by qNano (Izon Science) instrument, using the Tunable Resistive Pulse Sensing (TRPS) principle. This principle enables reception of signal while a single particle transfers through the NP150A membrane with pores of 150 nm. In order to eliminate contaminating debris, EV samples were passed through 0.22 μm filters. The apparatus was operated at a voltage of 0.48 V‐0.64 V and a pressure equivalent to 8 cm of H₂O. The membrane was stretched to 45‐47 mm. Polystyrene beads at a concentration of 1 × 10¹³ beads/mL (114 nm; CPC100 Izon Science) were used to calibrate size and concentration, following the manufacturer's instructions. Samples were diluted 1000‐fold with PBS buffer and measured over 10 minutes. The movement of the particle through the membrane was identified as change in the ionic stream causing voltage changes. The power of the signal is proportional to the particle size. According to the amount of particles and their velocity at a specific time, the qNano determines the EVs’ concentration.